Figure 6: CHK1-SSBP1 regulates mitochondrial translation to control mitochondrial/nuclear H₂O₂ levels.

(A) CHK1 regulates mitochondrial H₂O₂ levels in a SSBP1-dependent manner. H₂O₂ levels were determined with HyPer7 localized to the mitochondrial membrane in K562 cells depleted of the indicated genes. (B) Mitochondrial H₂O₂ levels regulate nuclear H₂O₂ levels. Nuclear H₂O₂ was measured with HyPer7 in K562 cells treated with mitoTEMPO and CHK1i. (C) Mitochondrial H₂O₂ increases CHK1i cytotoxicity. Proliferation was determined by measuring relative ATP levels. (D) Suppression of complex I (S1QEL1.1) but not complex III (S3QEL-2) superoxide partially reverts CHK1i-mediated nuclear H₂O₂. (E) CRISPRi scores of the indicated genes involved in mitochondrial translation. (F) CHK1 regulates mitochondrial translated proteins in a SSBP1-dependent manner. Immunoblot analysis of the indicated proteins in cells depleted of SSBP1 and treated with CHK1i. (G) CHK1-regulation of mitochondrial translation depends on SSPB1 localization. Immunoblot analysis of the indicated mitochondrial proteins as described in (F) in cells expressing SSBP1 phosphorylation mutants in K562 cells depleted of SSBP1. (H) Inhibition of CHK1 increases mitochondrial translation. Expression of MT-CO2 was determined by immunoblot and normalized to β-actin following CHK1i treatment (see also methods, Figure S6H). (I) Doxycycline (DOXY) decreases mitochondrially translated proteins. (J) DOXY treatment reduces CHK1i-mediated nuclear H₂O₂ levels. Scale Bar=10 μm. Data are represented as mean ± SEM. ***p< 0.0001. Statistical significance was determined by Student’s t-test (two-tailed, unpaired).